Phosphorothioate analogs of sn-2 radyl lysophosphatidic acid (LPA): Metabolically stabilized LPA receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2013.01.002

We describe an efficient synthesis of metabolically stabilized sn-2 radyl phosphorothioate analogs of lysophosphatidic acid (LPA), and the determination of the agonist activity of each analog for the six LPA receptors (LPA _1-6) using a recently developed TGFα shedding assay. In general, the sn-2 radyl OMPT analogs showed similar agonist activities to the previous 1-oleoyl-2-O-methyl-glycerophosphothioate (sn-1 OMPT) analogs for LPA_1-6 receptors. In most cases, the sn-2 radyl-OMPT analogs were more potent agonists than LPA itself. Most importantly, sn-2 alkyl OMPT analogs were very potent LPA₅ and LPA₆ agonists. The availability of sn-2 radyl OPMT analogs further refines the structure-activity relationships for ligand-receptor interactions for this class of GPCRs.

Full text of DOI:10.1016/j.bmcl.2013.01.002

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1865–1869
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Bioorganic & Medicinal Chemistry Letters
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Phosphorothioate analogs of sn-2 radyl lysophosphatidic acid (LPA):
Metabolically stabilized LPA receptor agonists
Guowei Jiang a, Asuka Inoue b, Junken Aoki b,c, Glenn D. Prestwich a,
⇑
a Department of Medicinal Chemistry, The University of Utah, 419 Wakara Way, Suite 205, Salt Lake City, UT 84108-1257, United States
b Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai 980-8578, Japan
c Japan Science and Technology Agency, Core Research for Evolutional Sciences and Technology (CREST), Kawaguchi, Saitama 332-0012, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe an efficient synthesis of metabolically stabilized sn-2 radyl phosphorothioate analogs of
Received 13 November 2012
Revised 29 December 2012
Accepted 2 January 2013
Available online 23 January 2013
lysophosphatidic acid (LPA), and the determination of the agonist activity of each analog for the six
LPA receptors (LPA1–6) using a recently developed TGFa shedding assay. In general, the sn-2 radyl OMPT
analogs showed similar agonist activities to the previous 1-oleoyl-2-O-methyl-glycerophosphothioate
(sn-1 OMPT) analogs for LPA1–6 receptors. In most cases, the sn-2 radyl-OMPT analogs were more potent
agonists than LPA itself. Most importantly, sn-2 alkyl OMPT analogs were very potent LPA5 and LPA6 ago-
nists. The availability of sn-2 radyl OPMT analogs further refines the structure–activity relationships for
ligand–receptor interactions for this class of GPCRs.
Keywords:
Lysophosphatidic acid
sn-2 Radyl phosphorothioate
Agonist
Ó 2013 Elsevier Ltd. All rights reserved.
Structure–activity relationship
Transforming growth factor-(TGF
shedding assay
a)
Lysophosphatidic acid (LPA, 1- or 2-radyl-sn-glycerol 3-phos-
phate) elicits growth-factor-like effects in variety of cell
tein family. So far, there are six different LPA GPCRs characterized
on the surface of mammalian cells in two distinct gene families.
The first three belong to endothelial differentiation genes (EDG),
EDG2/LPA1, EDG4/LPA2, and EDG7/LPA3.12 The second cluster of
a
types.1–4 Based on animal experiments, LPA is implicated in com-
plex physiological responses that include immunological compe-
tence, brain development, blood vessel formation, wound
healing, coagulation, embryo implantation and hair formation.5–7
The pleiotropic physiological functions of LPA suggest that LPA
could contribute to a number of pathophysiological states includ-
ing cancer, atherosclerosis, hypertension, ischemia reperfusion in-
jury, diabetes, cardiovascular diseases, stroke, prevention of
toxicity of chemotherapy and radiation therapy, immunomodula-
tion, and others.8
LPA GPCRs including GPR23/P2Y9/LPA4,13 GPR92/LPA5,14 and
15,16
P2Y5/LPA6
are members of the purinergic cluster in the GPCR
superfamily. LPA also activates the soluble nuclear peroxisome
proliferator-activated receptor
c
(PPAR
c
).17 Through activation of
O
P
OH
O
O
P
OH
HO
O
R
OH
R
O
O
OH
O
LPA can be generated either extracellularly or intracellularly in
response to various stimuli including growth factors, LPA itself,
phorbol esters, and epidermal growth factor (EGF). LPA is mainly
produced sequentially by two enzymatic reactions. In cellular
membrane, the action of membrane-associated secretory enzymes
phospholipase A1 or A2 on phosphatidic acid (PA) at either sn-1 or
sn-2 position gives lysophosphatidic acid.9 In extracellular fluids
such as serum, the lysophospholipase D (lysoPLD) activity of auto-
taxin (ATX) hydrolyzes lysophosphatidylcholine (LPC) to LPA.10,11
LPA signals through the activation of specific receptors which in
turn leads to distinct cellular events depending on the receptor
subtype expressed by the targeted cell. Cell-surface LPA receptors
belong to the membrane G-protein-coupled receptor (GPCR) pro-
OH
LPA
O
sn-2 LPA
O
S
P
OH
S
P
OH
C
17H33
O
O
OH
C17H33
1-
O
O
OH
OMe
OMe
1-oleoyl OMPT (2
R)
O
-oleyl OMPT (2R)
S
P
OH
O
S
P
C17H33
O
O
OH
C17H33
O
O
OH
OH
OMe
OMe
1-
O
-oleyl OMPT (2S)
1-oleoyl OMPT (2
S)
⇑
Corresponding author. Fax: +1 801 585 9053.
E-mail address: gprestwich@pharm.utah.edu (G.D. Prestwich).
Figure 1. LPA and its phosphorothioate analogs.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.002

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G. Jiang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1865–1869
S
P
S
P
S
P
ONa
MeO
O
C
ONa
ONa
MeO
O
MeO
O
OH
OH
C17H33
O
OH
C15H31
17H33
O
O
O
2
1
3
Figure 2. Phosphorothioate analogs of sn-2 radyl LPA.
involvement of the LPA–LPA6 axis in the regulation of hair follicle
development and hair growth. It is of interest to examine whether
LPA6 activation might induce hair growth. However, because LPA is
rapidly degraded in vivo, it is difficult to test the above-mentioned
notion. Hence, the development of more stable, potent agonists/
antagonists for emerging LPA receptors is eagerly awaited.
In general, the sn-2-substituted LPA analogs appeared more po-
tent than the corresponding sn-1 LPA analogs for activation of
LPA6.22 In addition, the phosphorothioate analogs of LPA, which
were previously reported as LPA3-selective agonist, was more po-
tent at the LPA6 receptor than LPA (18:1)23,24 (Fig. 1). Herein, we
describe the synthesis and pharmacology of three phosphorothio-
ate analogs of sn-2 radyl lysophosphatidic acid (Fig. 2). Interest-
ingly, except their agonist activities for LPA6 and LPA3, two alkyl
sn-2 phosphorothioate analogs exhibited most potent LPA5 activity
compared with previously identified LPA receptor agonists.
The synthetic strategies for the preparation of target phosp-
horothioate were based on two critical considerations. First, phos-
phoramidite chemistry was used for efficient phosphorylation of
the primary or secondary alcohols. The resulting phosphite tries-
ters could be oxidized with elemental sulfur in situ to yield the cor-
responding phosphorothioates. Second, the 2-cyanoethyl
phosphate protecting groups, which could be removed under mild
basic conditions, were key elements for the sn-2 radyl OPMT
synthesis.
As illustrated in Scheme 1, racemic solketal (dimethyl-1,3-
dioxolane-4-methanol) was converted to the common intermedi-
ate 7 was completed in three straightforward steps. Methylation
of 1,2-O-isopropylidene glycerol was performed with methyl io-
dide in DMF at room temperature to give methyl ether 5.
10 mol % p-TsOH in methanol was employed to remove the aceto-
nide to obtain the 1,2-diol 6 in 88% isolated yield. To minimize the
possible migration product from sn-2 to sn-1, a stable and easy to
remove protecting group should be elected for sn-1 hydroxyl; the
trityl (triphenylmethyl) group was used to protect the primary hy-
droxyl group (TrCl, Et3N) to give intermediate 7. The trityl group
could be cleaved by treating with trifluroacetic acid (TFA) at room
temperature for 1 h.
O
O
a
b
O
OMe
O
OH
5
4
OH
OH
Ph
Ph
O
OMe
c
HO
OMe
Ph
6
7
Scheme 1. Synthesis of common intermediate 7. Reagents and conditions: (a) MeI,
NaH, DMF, 5 h at room temperature, 75%; (b) PTSA, methanol, overnight at room
temperature, 88%; (c) TrCl, triethylamine, dichloromethane, overnight at room
temperature, 81%.
these GPCRs and PPAR
pathological responses.
c, LPA regulates multiple physiological and
GPR92/LPA5 is widely expressed in murine tissues such as
embryonic brain, small intestine, skin, spleen, stomach, thymus,
lung, heart, liver, and embryonic stem cells.18 Recent studies indi-
cated that two other lipid-derived molecules (farnesyl pyrophos-
phate and N-arachidonylglycine) were characterized as LPA5
ligands.19 However, following studies confirm that LPA5 is a bona
fide LPA receptor which can also be activated by farnesyl pyro-
phosphate at much higher concentrations compared to LPA
18:1.20 The biological function of the agonistic activity of alterna-
tive ligands is unclear yet. The studies reveal LPA5 may play an
important role in the response of neuropathic pain and human
platelet activation.21 Human P2Y5/LPA6 was originally reported
as an orphan GPCR sharing the highest sequence homology with
LPA4, which strongly indicated that LPA is a ligand for LPA6.15 In
a recent unbiased genetic search study, several mutations in the
LPAR6 gene (encoding LPA6) underlie autosomal recessive wooly/
sparse hair.15,16 We have recently shown that PA-PLA1
a/Liph is
responsible for LPA production and LPA6 activation during hair fol-
licle development in mice.7 These studies clearly revealed that the
OR
OH
OR
a
Ph
Ph
O
OMe
b
Ph
Ph
O
OMe
HO
OMe
Ph
Ph
9
7
8
9a
, R=C16H33
8a
, R=C16H33
9b, R=C18 35
H
8b, R=C18 35
H
CN
CN
OR
OR
OH
O ONa
O
P
S
d
c
MeO
MeO
O
O
P
S
10
1, R=C16H33
10a
, R=C16H33
2
, R=C18H35
10b, R=C18 35
H
Scheme 2. Synthesis of sn-2 alkyl phosphorothioate analogs 1 and 2. Reagents and conditions: (a) RI, NaH, DMF overnight at room temperature, 43–57%; (b) TFA, DCM, 1 h at
room temperature, 77–91%; (c) di-(2-cyanoethyl)-N,N-diisopropylphosphodiamidite, 1H-tetrazole; S, CS2, pyridine, two steps, 82–86%; (d) tBuNH2, BSA, 72 h, 67–84%.
BSA = N,O-bis(trimethylsilyl)acetamide, Dowex 50WX8-200 resin, neutral Na+ form.

G. Jiang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1865–1869
1867
O
O
OH
C17H33
Ph
Ph
O
C17H33
HO
O
a
b
Ph
Ph
O
OMe
O
OMe
OMe
Ph
Ph
7
12
11
O
O
CN
CN
C17H33
MeO
O
C17H33
MeO
O
OH
P
S
O
P
S
d
c
O
ONa
O
O
13
3
Scheme 3. Synthesis of sn-2 acyl phosphorothioate analogs 3. Reagents and conditions: (a) oleic acid, DCC, DMAP, DCM overnight at room temperature, 94%; (b) TFA, DCM,
1 h at room temperature, 81%; (c) di-(2-cyanoethyl)-N,N-diisopropylphosphodiamidite, 1H-tetrazole; S, CS2, pyridine, two steps, 67%; (d) tBuNH2, BSA, 72 h, Dowex 50WX8-
200 resin, neutral Na+ form, 81%.
With the compound 7 in hand, formation of ether to get inter-
mediate 8 was performed in the presence of sodium hydride
(Scheme 2). Then the trityl group was removed under trifluroacetic
acid (TFA) to provide alcohol 9, followed by phosphorylating with
phosphoramidite. The resulting phosphoramidite triester was oxi-
dized with elemental sulfur to obtain the corresponding phosp-
horothioate triester 10. In the penultimate step, cyanoethyl ester
groups were cleaved under aprotic basic conditions (tert-butyl
amine in acetonitrile) for 72 h to give the desired phosphorothioate
analog of LPA 1 and 2. The oily free acid forms were converted to
solid sodium salts by ion exchange.
two new compounds are most potent LPA5 agonists and could be
a useful pharmacological tool for the further study of ligand–recep-
tor interaction. The potent agonist activities of both sn-1 and sn-2
OMPT analogs for LPA5 could provide additional evidence that LPA5
is a true LPA receptors.19–23
In summary, we have developed a synthetic route for sn-2 radyl
OMPT by using phosphoramidite chemistry. Efforts to prepare the
enantiomers are in progress and will be reported in due course. The
racemic sn-2 alkyl OMPT analogs showed the most potent LPA5
In Scheme 3, acylation of sn-2 hydroxyl group with oleic acid
was conducted in dichloromethane in the presence of DCC and
DMAP to yield intermediate 11. The final target ester analog 3
was prepared from the precursor 11 in the analogous fashion to
that described previously.
The ligand properties of the compounds were evaluated using a
recently developed method to detect activation of GPCRs, namely
transforming growth factor-a (TGFa
) shedding assay25 for assess-
ing the activation of the six human LPA receptors (LPA1, LPA2,
LPA3, LPA4, LPA5, and LPA6) expressed in HEK293 cells. The assay
utilizes GPCR-induced ectodomain shedding of transmembrane
proform of TGF
with alkaline phosphatase (AP). After stimulation of LPA receptor,
cleaved AP-TGF is quantified by measuring AP activity in the con-
a. To quantify TGFa shedding easily, TGFa is fused
a
ditioned media using p-nitrophenyphosphate (p-NPP) as a sub-
strate. To gain maximal receptor-specific responses, we
employed following methods: co-expression of chimeric G
a pro-
teins24 (LPA1 and LPA5 with G
aq/i1; LPA2 and LPA4 with Gaq/s
)
and pretreatment with Ki16425, an LPA1–3 antagonist,26 (for
LPA4–6). Figure 3 and Table 1 illustrate LPA-induced response elic-
ited through the activation of human LPA1–6. Because HEK293 cells
endogenously express all the LPA receptors, we calculated param-
eters from transfected receptor–specific response (i.e., for each
dose, response in mock-transfected cells was subtracted from that
in receptor-transfected cells) (Fig. 3).
The two racemic OPMT analogs 2 and 3, both alkyl (18:1) and
acyl (18:1), are potent LPA3 agonists. Their potency (relative intrin-
sic activity (RIA)27 = 340 (2) and 600 (3); mean values) is compara-
ble to those of (2S) 1-acyl (RIA = 500) and (2S) 1-alkyl OMPT
(RIA = 390) analogs and much higher than that of parent LPA
(18:1). This trend is also exhibited for LPA6, which is consistent
with previous study7 and confirmed that both LPA3 and LPA6 prefer
unsaturated fatty acid. Meanwhile, for the receptor LPA6, com-
pounds 2 and 3 are almost 15-fold more potent compared to par-
ent LPA (18:1). The most significant activity observed, however,
was the discovery that both alkyl analogs 1 (RIA = 300) and 2
(RIA = 26) are LPA5 agonists. To the best of our knowledge, these
Figure 3. LPA-induced AP-TGF
HEK293 cells transfected with LPA receptor-encoding vector (receptor) or control
vector (mock) with or without chimeric G -encoding vector were subjected to the
TGF shedding assay. Note that LPA1 and LPA5 were co-expressed with Gaq/i1 and
LPA2 and LPA4 were co-expressed with G q/s. Also note that LPA4–6-expressing cells
were pretreated with 10 M Ki16425. Receptor-specific response, values of LPA
receptor-expressing cells subtracted from those of mock-transfected cells. Error
bars, SD for three to six assay replicates from one experiment. Data are represen-
tative of three experiments. For most data points, error bars are smaller than
symbols.
a release responses in the six LPA receptors (LPA1–6).
a
a
a
l

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G. Jiang et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1865–1869
Table 1
Pharmacological evaluation of the phosphorothioate analogs on the six LPA receptors in the TGF
a
shedding assay
LPA4
LPA1
LPA2
LPA3
LPA5
LPA6
pEC50 (M)
pEC50 (M)
(EC50 (nM))
[RIA]
pEC50 (M)
(EC50 (nM))
[RIA]
pEC50 (M)
(EC50 (nM))
[RIA]
pEC50 (M)
(EC50 (nM))
[RIA]
pEC50 (M)
(EC50 (nM))
[RIA]
a
(EC50 (nM))
[RIA]b
LPA (18:1)
LPA (16:0)
1-Oleoyl (2S)
1-Oleoyl (2R)
1-O-Oleyl (2S)
1-O-Oleyl (2R)
1
À7.75 0.10
(18)
[1]
À7.54 0.04
(29)
[1]
À7.11 0.09
(77)
[1]
À8.09 0.14
(8.1)
[1]
À7.26 0.08
(55)
[1]
À6.01 0.20
(970)
[1]
À7.15 0.12
À6.54 0.13
À6.24 0.10
À7.08 0.35
À7.17 0.06
À6.03 0.11
(71)
(290)
(580)
(84)
(68)
(930)
[0.24 0.02]
À7.83 0.09
(14.7)
[0.94 0.15]
À7.62 0.10
(24)
[0.61 0.15]
À7.99 0.10
(10.1)
[1.20 0.09]
À7.51 0.18
(31)
[0.092 0.023]
À6.95 0.08
(112)
[0.25 0.07]
À6.83 0.10
(149)
[0.178 0.033]
À6.96 0.20
(110)
[0.34 0.17]
À6.45 0.18
(350)
[0.102 0.036]
À9.57 0.20
(0.27)
[500 330]
À9.35 0.16
(0.44)
[260 150]
À9.43 0.24
(0.37)
[390 290]
À8.97 0.25
(1.08)
[0.14 0.04]
À8.57 0.21
(2.7)
[3.1 0.7]
À8.31 0.18
(4.9)
[1.6 0.2]
À8.46 0.12
(3.5)
[2.7 0.3]
À8.31 0.31
(4.9)
[1.04 0.31]
À8.30 0.16
(5.1)
[14.8 4.7]
À8.09 0.07
(8.2)
[8.4 1.2]
À8.47 0.23
(3.4)
[0.25 0.04]
À7.10 0.13
(79)
[13.1 3.3]
À7.23 0.08
(58)
[19.3 5.4]
À7.05 0.16
(90)
[11.0 2.1]
À6.98 0.18
(105)
[23 9]
À8.21 0.31
(6.1)
[0.57 0.17]
À7.58 0.04
(26)
[0.088 0.047]
À6.94 0.06
(116)
[140 100]
À9.44 0.12
(0.36)
[2.1 0.9]
À8.42 0.06
(3.8)
[16.0 10.0]
À9.59 0.22
(0.26)
[10.9 2.6]
À7.18 0.04
(66)
[0.38 0.06]
À7.95 0.12
(11.1)
[0.046 0.006]
À6.93 0.17
(117)
[300 150]
À9.40 0.20
(0.39)
[2.1 0.6]
À8.35 0.21
(4.5)
[300 120]
À8.52 0.22
(3.0)
[10.9 3.3]
À7.11 0.14
(78)
2
[1.29 0.10]
À7.91 0.11
(12.2)
[0.30 0.15]
À6.83 0.11
(146)
[340 230]
À9.69 0.17
(0.21)
[2.0 0.4]
À8.61 0.07
(2.4)
[26 11]
À8.74 0.16
(1.81)
[15.1 5.2]
À7.12 0.11
(76)
3
[1.15 0.12]
À7.78 0.10
(16.5)
[0.79 0.10]
À8.08 0.10
(8.4)
[0.165 0.05]
À6.79 0.12
(161)
[0.188 0.066]
À7.00 0.08
(100)
[600 360]
À9.56 0.15
(0.28)
[410 230]
À9.65 0.21
(0.23)
[3.9 1.5]
À8.48 0.21
(3.3)
[2.6 1.1]
À8.42 0.18
(3.8)
[41 15]
À8.20 0.15
(6.3)
[11.1 2.8]
À9.08 0.30
(0.83)
[14.3 4.2]
À7.07 0.08
(86)
[13.3 3.3]
À7.41 0.12
(39)
1-Oleoyl (2RS)
1-O-Oleyl (2RS)
[1.63 0.17]
[0.26 0.07]
[570 380]
[2.2 0.3]
[114 69]
[27 9]
Data are mean SEM (n = 3) for pEC50 and RIA.
a
EC50 values are calculated from mean pEC50 values.
RIA, relative intrinsic activity (Ref. 27), represents Emax/EC50 values relative to that of control ligand (here indicating LPA (18:1)).
b
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agonists compared to the agonists prepared before, which could be
used for the further pharmacological study.
Acknowledgments
J.A. was supported by Grants from the National Institute of Bio-
medical Innovation of Japan, the National Project on Protein Struc-
tural and Functional Analyses Grant-in-Aid for Scientific Research
on Innovative Areas (KAKENHI 22116004) from the Ministry of
Education, Science, Sports and Culture of Japan (MEXT) and Core
Research for Evolutional Sciences and Technology (CREST) from
Japan Science and Technology Agency. I.A. was funded by a
Grant-in-Aid for Young Scientists (B) (KAKENHI 21790058).
G.W.J. thanks Schering-Plough for support, and G.D.P. thanks the
University of Utah for support.
Supplementary data
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